Development of nano carriers to introduce nucleic acids into cells has been of considerable interest in biomedical research because of the potential of exogenously delivered therapeutic nucleic acids to cure several genetic as well as complex disorders [Edelstein et al., 2007]. Viral vectors are most explored in this context but have been found in many cases to be unsafe for clinical use because of associated immune response and random integration in host genome. A variety of materials (cationic lipids and polymers) have been used to facilitate the delivery of nucleic acids into the cells [Meredith et al (2009), Niidome et al (2002)]. But most of these materials have limitations like low transfection efficiency, high cytotoxicity and complicated synthesis procedure which leads to limited product yield [Mintzer et. al., 2009; Remaut et. al., 2007]. For example, although liposomes are attractive materials for drug delivery applications, application of liposomes for DNA delivery is plagued by problems like cytotoxicity, serum instability, changes in size, surface charge and lipid composition of the lipid-DNA complex during delivery and so on (Liu et al., 2003). Cationic polymers have been used as a substitute of lipid based vectors but most polymers also exhibit some major disadvantages, e.g. complicated synthetic procedure, lack of control during synthesis which ultimately reflects in non-uniform physico-chemical properties, low biodegradability and high toxicity. Besides there are difficulties of selectively modifying the polymer with ligands for targeted delivery. One of the cationic polymers extensively used for gene delivery, poly (ethyleneimine) (PEI), suffers due to its non-biodegradable nature leading to cellular toxicity (Fischer et al., 2003).
Peptides constitute a promising class of non-viral vectors as they are easy to synthesize, amenable to modifications for the attachment of different functional moieties, relatively small in size and are the most biocompatible class of delivery vectors [Fabre et al 2006]. Peptides used for nucleic acid delivery can be categorized into two classes: cationic peptides and amphipathic peptides. Amphipathic peptides are made of both hydrophobic and hydrophilic amino acids [Fernandez-Carneado, 2004]. The basic framework of the cell membrane is made up of amphipathic lipids; hence amphipathic peptides can interact with the membrane in a more efficient manner and allow cargo uptake [Fomiyana et al., 2000; Kuriyama et al., 2006; Bartz et al., 2011; Oehlke et al., 2004; Niidome et al., 2000]. Therefore naturally occurring as well as synthetically designed amphipathic peptides are of great interest as delivery vectors [Elmquist et al (2001) and Wyman et al (1997)]. Most amphipathic peptides possess the ability to translocate across the cell membrane. Efficient cellular uptake and endosomal escape properties of these peptides are the main reasons for their development as carriers of therapeutic cargo molecules. However, most of these peptides are not effective carriers of large cargoes like plasmid DNA and it is important to optimally design the peptide to improve the delivery efficiency [Rajpal et al (2012)]. The hydrophobicity and positive charge are two most important parameters of the peptide which need to be optimized for designing an amphipathic peptide which can deliver plasmid DNA with high efficiency through formation of a nanocomplex. The hydrophobic amino acids of the peptide interact with membrane and help in cellular uptake of the nanocomplex and positive charged amino acids of the peptide condense DNA to form these nano complexes. A minimum amount of each of these two types of amino acids is required; however the proportion of these two residues should be optimized because high hydrophobicity as well as positive charge (arginine) can cause cellular toxicity, and tight packaging of DNA reduces the accessibility of DNA to transcription machinery [Mann et al (2011)]. On the other hand, less number of positive charges and low hydrophobicity can lead to premature degradation of the DNA and poor cellular uptake [Niidome et al (1997) and Niidome et al (1999)].
In the present invention, nanocomplex has been prepared containing Mgpe peptides. The peptide Mgpe-1 (SEQ ID NO:2) (derived from Human Protein phosphatase 1E (SEQ ID NO:1) is a novel amphipathic peptide for biomolecule delivery and we further altered its physicochemical parameters and generated five novel peptides to achieve efficient delivery of plasmid DNA and small nucleic acid. The primary sequence of Mgpe-1 peptide is SRLSHLRHHYSKKWHRFR (Mgpe1) (SEQ ID NO:2). In a modification of Mgpe-1 (SEQ ID NO:2) total charges have been increased from 6 to 9 and Mgpe-3 has been generated (RRLRHLRHHYRRRWHRFR) (SEQ ID NO:3). Further, a peptide Mgpe-4 (LLYWFRRRHRHHRRRHRR) (SEQ ID NO:4) has been generated by altering the amphipathicity of Mgpe-3 from secondary to primary by altering the position of amino acids. These two peptides (Mgpe-3 and Mgpe-4) were further modified by addition of two cysteine residues at both ends. These two primary sequences are CRRLRHLRHHYRRRWHRFRC and CLLYWFRRRHRHHRRRHRRC (Mgpe 9 and Mgpe 10, SEQ ID Nos. 5 and 6, respectively). All modified Mgpe peptides were generated in such a way as to optimize the total content of hydrophobicity, charges, amphipathicity and amino acid composition and were developed by modifying the primary sequence of the Mgpe-1 peptide (SEQ ID NO:2) sequentially. All the peptides formed nanocomplexes with plasmid DNA with particle sizes 50-110 nm and exhibited high transfection efficiency in multiple cell lines with negligible or very low toxicity.